Proteomic and transcriptomic study on the action of a cytotoxic saponin (Polyphyllin D): Induction of endoplasmic reticulum stress and mitochondria‐mediated apoptotic pathways

Polyphyllin D (PD) is a potent cytotoxic saponin found in Paris polyphylla. In the present study, bioinformatic, proteomic and transcriptomic analyses were performed to study the mechanisms of action of PD on human nonsmall cell lung cancer (NSCLC) cell line (NCI‐H460). Using a gene expression‐based bioinformatic tool (connectivity map), PD was identified as a potential ER stress inducer. Our proteomic and transcriptomic analyses revealed that PD treatment led to upregulation of typical ER stress‐related proteins/genes including glucose‐regulated protein 78 (BiP/GRP78) and protein disulfide isomerase (PDI). In particular, elevated expression of C/EBP homologous transcription factor (chop) and activation of caspase‐4 occurred at early time point (8 h) of PD treatment, signifying an initial ER stress‐mediated apoptosis. Induction of tumor suppressor p53, disruption of mitochondrial membrane, activation of caspase‐9 and caspase‐3 were detected upon prolonged PD treatment. Collectively, these data revealed that PD induced the cytotoxic effect through a mechanism initiated by ER stress followed by mitochondrial apoptotic pathway. The ability of activating two major pathways of apoptosis makes PD an attractive drug lead for anticancer therapeutics.

[1]  Hong Wang,et al.  Endoplasmic Reticulum Stress Stimulates Heme Oxygenase-1 Gene Expression in Vascular Smooth Muscle , 2005, Journal of Biological Chemistry.

[2]  P. Sutton,et al.  Saponin-adjuvanted particulate vaccines for clinical use. , 2006, Methods.

[3]  Tsonwin Hai,et al.  Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response , 2004, Molecular and Cellular Biology.

[4]  Ca2+-induced redistribution of Ca2+/calmodulin-dependent protein kinase II associated with an endoplasmic reticulum stress response in vascular smooth muscle , 2000, Molecular and Cellular Biochemistry.

[5]  R. Kaufman,et al.  From acute ER stress to physiological roles of the Unfolded Protein Response , 2006, Cell Death and Differentiation.

[6]  Biao Yu,et al.  Effects of polyphyllin D, a steroidal saponin in Paris Polyphylla, in growth inhibition of human breast cancer cells and in xenograft , 2005, Cancer biology & therapy.

[7]  M. Pino,et al.  Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis. , 2005, Cancer research.

[8]  Hanry Yu,et al.  Kinectin-dependent Assembly of Translation Elongation Factor-1 Complex on Endoplasmic Reticulum Regulates Protein Synthesis* , 2006, Journal of Biological Chemistry.

[9]  R. Noiva.  Protein disulfide isomerase: the multifunctional redox chaperone of the endoplasmic reticulum. , 1999, Seminars in cell & developmental biology.

[10]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[11]  Jiangbing Zhou,et al.  The mitotic-arresting and apoptosis-inducing effects of diosgenyl saponins on human leukemia cell lines. , 2004, Biological & pharmaceutical bulletin.

[12]  T. Kudo,et al.  Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Aβ-induced cell death , 2004, The Journal of cell biology.

[13]  O. Brodin,et al.  Chemotherapy in non-small cell lung cancer: a meta-analysis using updated data on individual patients from 52 randomised clinical trials , 1995 .

[14]  Hong‐Tai Chang,et al.  Effect of calmidazolium on Ca2+ movement and proliferation in human osteosarcoma cells , 2004 .

[15]  Tsonwin Hai,et al.  Activating transcription factor 3, a stress sensor, activates p53 by blocking its ubiquitination , 2005, The EMBO journal.

[16]  A. Goldin,et al.  Effects of glucose analogues (2-deoxy-D-glucose, 2-deoxy-D-galactose) on experimental tumors. , 1960, Journal of the National Cancer Institute.

[17]  C. Conn,et al.  The complete primary structure of mouse 20S proteasomes , 1999, Immunogenetics.

[18]  H. Hwang,et al.  Proteomic analysis for inhibitory effect of chitosan oligosaccharides on 3T3‐L1 adipocyte differentiation , 2008, Proteomics.

[19]  M. Brostrom,et al.  Inhibition of protein synthesis and early protein processing by thapsigargin in cultured cells. , 1993, The Biochemical journal.

[20]  D. Bredesen,et al.  Coupling endoplasmic reticulum stress to the cell death program , 2004, Cell Death and Differentiation.

[21]  G. Shore,et al.  Regulation of apoptosis by endoplasmic reticulum pathways , 2003, Oncogene.

[22]  S. Korsmeyer,et al.  Proapoptotic BAX and BAK: A Requisite Gateway to Mitochondrial Dysfunction and Death , 2001, Science.

[23]  L. Hendershot,et al.  Geldanamycin, an hsp90/GRP94‐binding drug, induces increased transcription of endoplasmic reticulum (ER) chaperones via the ER stress pathway , 1998, Journal of cellular physiology.

[24]  J. Halperin,et al.  Depletion of intracellular Ca2+ stores, phosphorylation of eIF2alpha, and sustained inhibition of translation initiation mediate the anticancer effects of clotrimazole. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[25]  D. Bredesen,et al.  Coupling Endoplasmic Reticulum Stress to the Cell Death Program , 2002, The Journal of Biological Chemistry.

[26]  Qing‐Yu He,et al.  Dioscin (saponin)-induced generation of reactive oxygen species through mitochondria dysfunction: a proteomic-based study. , 2007, Journal of proteome research.

[27]  Yi-Ching Lee,et al.  Endoplasmic reticulum stress-induced caspase-4 activation mediates apoptosis and neurodegeneration in INCL. , 2006, Human Molecular Genetics.

[28]  John Calvin Reed,et al.  Identification of a p53-dependent negative response element in the bcl-2 gene. , 1994, Cancer research.

[29]  Johan Hansson,et al.  Cisplatin Induces Endoplasmic Reticulum Stress and Nucleus-independent Apoptotic Signaling* , 2003, The Journal of Biological Chemistry.

[30]  Paul A Clemons,et al.  The Connectivity Map: Using Gene-Expression Signatures to Connect Small Molecules, Genes, and Disease , 2006, Science.

[31]  R. Flavell,et al.  Endoplasmic Reticulum Stress-induced Death of Mouse Embryonic Fibroblasts Requires the Intrinsic Pathway of Apoptosis* , 2007, Journal of Biological Chemistry.

[32]  M. Michaelis,et al.  beta-Amyloid and endoplasmic reticulum stress responses in primary neurons: effects of drugs that interact with the cytoskeleton. , 2006, Journal of molecular neuroscience : MN.

[33]  A. Rao,et al.  Saponins as anticarcinogens. , 1995, The Journal of nutrition.

[34]  T. Aw,et al.  Gadd153 Sensitizes Cells to Endoplasmic Reticulum Stress by Down-Regulating Bcl2 and Perturbing the Cellular Redox State , 2001, Molecular and Cellular Biology.

[35]  Biao Yu,et al.  The Cytotoxicity of Saponins Correlates with Their Cellular Internalization , 2007, ChemMedChem.

[36]  Neal N. Iwakoshi,et al.  Proteasome inhibitors disrupt the unfolded protein response in myeloma cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[37]  M. Kilberg,et al.  Amino Acid Deprivation and Endoplasmic Reticulum Stress Induce Expression of Multiple Activating Transcription Factor-3 mRNA Species That, When Overexpressed in HepG2 Cells, Modulate Transcription by the Human Asparagine Synthetase Promoter* , 2003, Journal of Biological Chemistry.

[38]  G. Scagliotti,et al.  Multidrug resistance in non-small-cell lung cancer. , 1999, Annals of oncology : official journal of the European Society for Medical Oncology.

[39]  Fengqin Gao,et al.  Bcl2's Flexible Loop Domain Regulates p53 Binding and Survival , 2006, Molecular and Cellular Biology.

[40]  Chu-Wei Kuo,et al.  A transcriptomic and proteomic analysis of the effect of CpG‐ODN on human THP‐1 monocytic leukemia cells , 2005, Proteomics.

[41]  A. Fienberg,et al.  , Isabella Response with Cytoprotective Properties Target of the Mammalian Unfolded Protein Characterization of Stanniocalcin 2 , a Novel , 2004 .

[42]  L. Ellerby,et al.  Coupling endoplasmic reticulum stress to the cell death program: role of the ER chaperone GRP78 , 2002, FEBS letters.

[43]  Y. H. Kim,et al.  ER stress-induced caspase-12 activation is inhibited by PKC in neuronal cells , 2005, Apoptosis.

[44]  Qing‐Yu He,et al.  Proteomic approach to study the cytotoxicity of dioscin (saponin) , 2006, Proteomics.

[45]  Po-Cheng Chiang,et al.  Genistein induces apoptosis in human hepatocellular carcinomas via interaction of endoplasmic reticulum stress and mitochondrial insult. , 2007, Biochemical pharmacology.

[46]  N. Brown,et al.  Studies of the active site of m-calpain and the interaction with calpastatin. , 1993, The Biochemical journal.

[47]  Di Chen,et al.  Celastrol, a triterpene extracted from the Chinese "Thunder of God Vine," is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. , 2006, Cancer research.

[48]  J. van Staden,et al.  Biological activities and distribution of plant saponins. , 2004, Journal of ethnopharmacology.

[49]  D. Xie,et al.  Clusterin plays an important role in hepatocellular carcinoma metastasis , 2006, Oncogene.

[50]  C. Czupalla,et al.  Comparative study of protein and mRNA expression during osteoclastogenesis , 2005, Proteomics.

[51]  K. Mori.  Tripartite Management of Unfolded Proteins in the Endoplasmic Reticulum , 2000, Cell.

[52]  X. Leighton,et al.  Annexin 7 mobilizes calcium from endoplasmic reticulum stores in brain. , 2004, Biochimica et biophysica acta.

[53]  S. Kong,et al.  Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells. , 2005, Cancer letters.

[54]  Amy S. Lee,et al.  Endoplasmic Reticulum Stress-induced Apoptosis , 2006, Journal of Biological Chemistry.

[55]  Zenon Grabarek,et al.  Structural basis for diversity of the EF-hand calcium-binding proteins. , 2006, Journal of molecular biology.

[56]  K. Fung,et al.  An improved synthesis of the saponin, polyphyllin D. , 2001, Carbohydrate Research.

[57]  J. Boelens,et al.  The Endoplasmic Reticulum: A Target for New Anticancer Drugs , 2007 .

[58]  S. Linder,et al.  Lysosomes and endoplasmic reticulum: targets for improved, selective anticancer therapy. , 2005, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[59]  S. Oyadomari,et al.  Roles of CHOP/GADD153 in endoplasmic reticulum stress , 2004, Cell Death and Differentiation.

[60]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.

[61]  C. Che,et al.  Fluorophore-appended steroidal saponin (dioscin and polyphyllin D) derivatives. , 2005, Organic letters.

[62]  D. Wolf,et al.  ER Degradation of a Misfolded Luminal Protein by the Cytosolic Ubiquitin-Proteasome Pathway , 1996, Science.

[63]  T. Momoi.  Caspases involved in ER stress-mediated cell death , 2004, Journal of Chemical Neuroanatomy.